PCR detection primers and detection method for banana gray streak pathogen

Abstract

The present invention provides a PCR detection primer and a detection method for banana gray streak pathogen, wherein the upstream primer sequence in the PCR detection primer is ATGTGCAAGGCCGGTTTCG, and the downstream primer sequence is CGTCACATCGGCTCAAATCG. Based on the specific primer, a PCR detection method for banana gray streak pathogen is developed. The primer and the detection method are used to quickly detect banana gray streak pathogen, and pathogens can be distinguished by a single PCR reaction combined with sequencing analysis, thereby overcoming the shortcomings of conventional identification methods, and having the advantages of strong specificity, high accuracy, high sensitivity, wide applicability, and simple operation.

Description

Technical Field

[0001] The invention relates to the technical field of crop fungal disease detection, and in particular to a PCR detection primer and a detection method for banana gray streak pathogen. Background Art

[0002] Banana (Musa spp.) is an important herbaceous fruit tree and food crop in tropical and subtropical regions. With the increase in the scale and intensiveness of banana production, the occurrence of diseases has become increasingly serious, which has had a serious impact on banana yield and quality. Cordana leaf spot of banana, also known as banana dark bisporus leaf spot, mainly harms banana leaves and is common in banana producing areas around the world. In the early stage of the disease, water-soaked, nearly circular or irregular light brown spots appear on the front of the leaves. After expansion, the spots become oval or fusiform, with light gray in the center, concentric rings, dark brown edges, and yellow halos at the junction of diseased and healthy leaves; in severe cases, the spots expand along the leaf margins and merge into large spots parallel to the midrib, with wavy and dry leaf margins and bright yellow halos on the periphery; when the field humidity is high, the spots on the back of the leaves are gray-brown and produce gray mold-like substances, namely the conidiophores and conidia of the pathogen. In production, the disease often occurs together with banana brown gray spot, coal leaf spot, crescent spore leaf spot, and coryneformis leaf spot. In severe cases, it can cause the whole leaf to dry up, the plant to age prematurely, and the fruit yield and quality to decrease. In recent years, banana gray leaf spot has been common in my country's banana producing areas and has become increasingly serious. It has become one of the common and serious diseases in my country's banana producing areas. Therefore, rapid detection of banana gray leaf spot pathogens is of great significance for early diagnosis and timely and effective prevention and control of the disease.

[0003] The pathogen of banana gray streak (Neocordana musae (Zimm.) Hern.-Restr. & Crous, synonym: Cordanamuase Zimm.) grows slowly on common culture media such as PDA, PSA, and OA. The colonies are easily covered by other bacteria during isolation, and the conidia are produced late, making it difficult to identify in the early stage. In addition, the conventional disease diagnosis technology based on symptoms requires Koch's postulates such as pathogen isolation and culture, morphological characteristics observation, pathogenicity test and symptom analysis, which takes a long time, and its accuracy and reliability are largely determined by the experience and taxonomic knowledge of the identifier; secondly, the disease often occurs in combination with other banana leaf diseases, making it difficult to detect the disease in time and effectively control the spread of the disease when it first occurs, which is difficult to meet the actual needs of banana gray streak disease diagnosis. Therefore, it is urgent to establish a set of rapid diagnosis technologies that are convenient, fast, reliable and highly sensitive.

[0004] In recent years, with the rapid development and application of modern molecular biotechnology, some molecular marker technologies have provided new ways for the diagnosis and detection of plant pathogens. Among them, PCR (polymerase chain reaction) technology has attracted attention for its rapid, specific and sensitive detection and identification of pure culture strains and samples with bacteria, and has been widely used in the detection of plant pathogens. At present, researchers at home and abroad have successfully developed specific primers and detection methods for banana brown gray spot fungus, banana anthracnose fungus, banana black spot fungus, banana crown rot fungus and other banana pathogens, achieving rapid, sensitive and accurate identification. However, there are no reports on the molecular detection of banana gray streak fungus. Summary of the invention

[0005] The existing technology for detecting and identifying banana gray streak pathogens is mainly based on the morphological characteristics of the pathogens. However, since the banana gray streak pathogens grow slowly, produce spores late, the isolation procedure is cumbersome and time-consuming, and their accuracy and reliability are largely determined by the experience and taxonomic knowledge of the identifier, it is difficult to meet the actual needs of banana gray streak disease diagnosis. The present invention provides PCR detection primers and a detection method specifically for banana gray streak disease pathogens, so as to realize rapid, sensitive and accurate identification of banana gray streak disease in the field.

[0006] The technical solution of the present invention mainly includes the following contents:

[0007] A PCR detection primer NMF / NMR2 for banana gray streak pathogen Neocordana musae, wherein the upstream primer sequence is ATGTGCAAGGCCGGTTTCG, and the downstream primer sequence is CGTCACATCGGCTCAAATCG.

[0008] The invention relates to application of the PCR detection primer NMF / NMR2 in detecting banana gray streak pathogen.

[0009] Based on the above-mentioned specific detection primers NMF / NMR2, the present invention further provides a PCR detection method for banana gray streak pathogen, comprising the following steps:

[0010] (1) Extracting genomic DNA from samples to be tested;

[0011] (2) Using the extracted genomic DNA of the sample to be tested as a template and NMF / NMR2 as primers, PCR amplification was performed;

[0012] (3) The PCR amplification product is subjected to electrophoresis analysis, and the detection result is determined according to the size of the amplified product; if a 268 bp product can be specifically amplified, it can be determined that the sample carries banana gray streak pathogen, otherwise, banana gray streak pathogen is not present in the sample.

[0013] Preferably, the annealing temperature of the PCR amplification is 55°C.

[0014] Preferably, the genomic DNA of the sample to be tested includes genomic DNA of pathogens or genomic DNA of banana leaves.

[0015] Preferably, the PCR reaction system is: 12.5 μL Premix Taq, 0.5 μL of 10 μmol / L upstream primer, 0.5 μL of 10 μmol / L downstream primer, 1 μL DNA template, supplemented with ddH2O to 25 μL.

[0016] Preferably, the PCR amplification reaction conditions are: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 45 sec, for a total of 30 cycles; extension at 72°C for 5 min; and storage at 10°C.

[0017] Compared with the prior art, the present invention has the following remarkable effects:

[0018] (1) Strong specificity: The primers NMF / NMR2 designed in the present invention have strong specificity to banana gray streak pathogen, and can be used to distinguish common banana pathogens such as banana coal streak pathogen, banana brown edge gray spot pathogen, banana black spot pathogen, banana crescent spore, banana alternator, banana pear spore, banana anthracnose fungus, banana coryneformis, banana wilt pathogen, etc., thereby effectively distinguishing diseases with similar symptoms occurring on banana.

[0019] (2) High accuracy: The present invention uses PCR primers NMF / NMR2 that have a specific amplification effect on banana gray streak pathogen, and conducts detection and verification on banana gray streak pathogen, other pathogens that harm bananas, banana leaves with typical symptoms of banana gray streak pathogen, artificially inoculated banana leaves, banana leaves mixed with banana gray streak pathogen and other banana leaf diseases, and healthy banana leaves. Only banana gray streak pathogen and banana leaf tissues carrying the pathogen can specifically amplify a 268 bp electrophoresis band, indicating that the primers NMF / NMR2 and the detection method designed by the present invention are accurate and reliable for detecting banana gray streak pathogen.

[0020] (3) High sensitivity: After the designed specific primers NMF / NMR2 are used for PCR amplification, the detection sensitivity of banana gray streak pathogen can reach 20 pg / μL at the DNA level.

[0021] (4) Wide applicability: The method for detecting banana gray streak pathogen of the present invention can not only detect the mycelium of the pathogen, but also detect the diseased banana leaves, and can realize the early detection of banana gray streak pathogen, that is, detection before the disease symptoms appear, which is of great significance for monitoring and preventing the outbreak of banana gray streak disease during the growth period.

[0022] (5) Easy operation: The present invention only requires DNA extraction, PCR amplification, agarose electrophoresis, and sequencing analysis of the amplified product to determine the result. Generally, the entire detection process can be completed within a few hours, and the operation is simple and fast. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :1% agarose gel electrophoresis of genomic DNA of some test samples. Lane M is Marker DL 5 000, lanes 1-5 are banana genomic DNA, and lanes 6-11 are banana gray streak pathogen genomic DNA.

[0024] Figure 2 :Electrophoregram of the effectiveness test of candidate primers against banana gray streak pathogen. AD are NMF / NMR1, NMF / NMR2, NMF / NMR3, NMF / NMR4, lane M is Marker DL2 000; lanes 1-3 are banana gray streak pathogen, and lane 4 is a negative control.

[0025] Figure 3 :The electrophoresis diagram of the specificity test of candidate primers to banana gray streak pathogen. AD are NMF / NMR1, NMF / NMR2, NMF / NMR3, and NMF / NMR4, respectively. Lane M is Marker DL2 000. Lanes 1-3 are banana gray streak pathogen, and lanes 4-11 are banana coal streak pathogen, banana brown edge gray spot pathogen, banana black spot pathogen, banana crescent spore, banana alternaria, banana pear spore, banana anthracnose pathogen, and negative control.

[0026] Figure 4 :The electrophoresis diagram of the sensitivity test of candidate primers to banana gray streak pathogen. Lanes AD are NMF / NMR1, NMF / NMR2, NMF / NMR3, and NMF / NMR4, respectively. Lane M is Marker DL2 000. Lanes 1-10 are 200ng / μL, 20ng / μL, 2ng / μL, 200pg / μL, 20pg / μL, 2pg / μL, 200fg / μL, 20fg / μL, 2fg / μL, and negative control, respectively.

[0027] Figure 5 : Electrophoresis of specific amplification of banana gray streak pathogen by specific primer NMF / NMR2. Lane M is MarkerDL2 000, lanes 1-3 are banana gray streak pathogen, lanes 4-13 are banana coal streak pathogen, banana brown edge gray spot pathogen, banana black spot pathogen, banana crescent spore fungus, banana alternator spore fungus, banana pear spore fungus, banana anthracnose fungus, banana coryneformis, banana wilt pathogen, and negative control.

[0028] Figure 6:The electrophoresis diagram of sensitivity detection of banana gray streak pathogen by specific primer NMF / NMR2. Lane M is MarkerDL2 000, and lanes 1-11 are 200ng / μL, 20ng / μL, 2ng / μL, 200pg / μL, 20pg / μL, 2pg / μL, 200fg / μL, 20fg / μL, 2fg / μL, and negative control, respectively.

[0029] Figure 7 :The electrophoresis diagram of the specific primer NMF / NMR2 detecting the gray streak pathogen of banana leaf tissue. Lane M is MarkerDL2000, and lanes 1-11 are respectively positive control, naturally diseased banana fresh leaves, naturally diseased banana old leaves, artificially inoculated symptomatic banana leaves, artificially inoculated non-symptomatic banana leaves, banana leaves mixed with banana gray streak and banana coal streak, banana leaves mixed with banana gray streak and banana crescent spore leaf spot, banana leaves mixed with banana gray streak and banana brown edge gray spot, banana leaves mixed with banana gray streak and banana coryneformis leaf spot, healthy banana leaves, and negative control. DETAILED DESCRIPTION

[0030] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0031] Example:

[0032] Step 1: Extraction of genomic DNA from samples to be tested

[0033] 1. CTAB method was used to extract pathogen pure culture hyphae and banana leaf tissue genomic DNA. The specific steps are as follows:

[0034] (1) Grind the sample thoroughly with liquid nitrogen and transfer the dry powder into a sterile centrifuge tube. Add CTAB buffer (20 g CTAB / L, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM EDTA, pH 8.0; 2% β-mercaptoethanol) at a ratio of 200 mg to 750 μL. Vortex to mix. Incubate in a 65°C water bath for 30 to 60 min, mixing by inverting every 10 min.

[0035] (2) Remove the centrifuge tube treated in a 65°C water bath in step (1) from the water bath, cool to room temperature, add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), and vortex to mix; centrifuge at 12,000 rpm for 10 min at room temperature.

[0036] (3) Take the supernatant after centrifugation in step (2) and transfer it to a new sterile centrifuge tube. Add an equal volume of chloroform:isoamyl alcohol (24:1) and vortex to mix. Centrifuge at room temperature and 12,000 rpm for 10 min. Repeat this step until the interface between the lower organic phase and the upper aqueous phase is clear.

[0037] (4) Take the supernatant after centrifugation in step (3) and transfer it to a new sterile centrifuge tube. Add 0.6 times the volume of isopropanol (precooled at -20°C), mix thoroughly, and place in a -20°C refrigerator for more than 30 minutes to precipitate DNA.

[0038] (5) taking out the centrifuge tube from the -20°C refrigerator in step (4), centrifuging at 4°C, 12,000 rpm for 10 min, and collecting the precipitated DNA;

[0039] (6) Discard the supernatant after centrifugation in step (5), add 750 μL of 70% ethanol, invert the centrifuge tube back and forth to wash the precipitate; centrifuge at 4°C, 12,000 rpm for 10 min, pour off the ethanol, invert the tube on clean absorbent paper, and dry the DNA precipitate; repeat this step once.

[0040] (7) Dissolve the DNA that has been fully dried in step (6) in 100 uL of sterile deionized water preheated to 55-65°C.

[0041] (8) The DNA dissolved in sterile deionized water in step (7) was subjected to electrophoresis on a 1% agarose gel. Figure 1 ) and stored in a -20℃ refrigerator.

[0042] Step 2: Sequence amplification and sequencing of target genes of banana gray streak pathogen

[0043] The genomic DNA of 23 banana gray streak pathogens stored in our laboratory was extracted by CTAB method. Primers ACT-512F (5'-ATGTGCAAGGCCGGTTTCGC-3') and ACT-783R (5'-TACGAGTCCTTCTGGCCCAT-3') were diluted to 10μmol / L, and PCR amplification was performed using the extracted genomic DNA of 23 banana gray streak pathogens as templates and sterile deionized water as negative control. The PCR amplification reaction system was: 12.5μL Premix Taq (Takara Dalian Bao Biotechnology Co., Ltd.), 0.5μL of 10μmol / L primers (ACT-512F and ACT-783R), 1μL DNA template, and ddH2O was added to 25μL. The PCR amplification reaction conditions were: 95℃ pre-denaturation for 5min; 95℃ denaturation for 1min, 58℃ annealing for 1min, 72℃ extension for 1min, a total of 30 cycles; 72℃ extension for 8min; 10℃ storage. After the PCR reaction was completed, 5 μL of the reaction product was taken for 1% agarose gel electrophoresis analysis, and the remaining PCR amplification product was sent to a sequencing company (Shenzhen BGI Genomics Co., Ltd.) for sequencing.

[0044] Step 3. Design and screening of specific primers

[0045] 1. Primer Design and Synthesis

[0046] Primers ACT-512F and ACT-783R were used to amplify and sequence the genomic DNA of 23 banana gray streak pathogens by PCR, and 23 sequence information was obtained, with a similarity of 100%; 4 pairs of primers (synthesized by Shenzhen BGI Technology Co., Ltd.) were designed with the obtained 23 sequences as targets. The primer sequences and amplified fragment sizes are shown in Table 1.

[0047] Table 1 Four pairs of primers and sequences designed to detect banana gray streak pathogen

[0048]

[0049] 2. Primer screening

[0050] 2.1 Validity test The designed and synthesized primers (Table 1) were diluted to 10 μmol / L, and the genomic DNA of three strains of banana gray streak pathogen was extracted as template, and sterile deionized water was used as negative control. Four annealing temperatures (53, 55, 57, and 60°C) were set for PCR amplification to verify whether the designed primers were usable. The PCR amplification reaction system was: 12.5 μL Premix Taq (Takara Dalian Bao Biotechnology Co., Ltd.), 0.5 μL of each 10 μmol / L primer, 1 μL DNA template, and ddH2O supplemented to 25 μL. The PCR amplification reaction conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 sec, 53, 55, 57, and 60°C annealing for 30 sec, 72°C extension for 45 sec, a total of 30 cycles; 72°C extension for 5 min; 10°C storage. After the PCR reaction was completed, 5 μL of the reaction product was taken for 1% agarose gel electrophoresis analysis. It was found that at 53, 55, 57, and 60°C annealing temperatures, the four pairs of primers could effectively amplify the test strains and obtain target fragments of 235 bp, 268 bp, 299 bp, and 306 bp, respectively. Among them, the amplification band at 55°C was the brightest and had the highest amplification efficiency. Figure 2 As shown in the figure. At 55°C annealing temperature, the amplified band of primer NMF / NMR4 was relatively thin and the product amount was relatively small. The amplified products of primers NMF / NMR1, NMF / NMR2, and NMF / NMR3 were relatively large.

[0051] 2.2 Specificity test

[0052] Three strains of banana gray streak pathogen and seven strains of other banana pathogens were selected to test the specificity of four pairs of primers (Table 1), and the primers that can amplify banana gray streak pathogen strains but cannot amplify other strains were screened. The CTAB method was used to extract the genomic DNA of banana gray streak pathogen, banana coal streak pathogen, banana brown-margin gray spot pathogen, banana black spot pathogen, banana crescent spore, banana alternaria, banana pear spore, and banana anthracnose fungus. Using the extracted genomic DNA of the test strain as a template and sterile deionized water as a negative control, PCR amplification was performed at an annealing temperature of 55°C and referring to the validity method in 2.1. After the PCR reaction was completed, 5 μL of the reaction product was subjected to 1% agarose gel electrophoresis. Figure 3 As shown in the figure, the four pairs of primers amplified single target fragments (AD) of 235 bp, 268 bp, 299 bp and 306 bp respectively from the three strains of banana gray streak pathogen, among which primers NMF / NMR2 and NMF / NMR4 could not amplify bands (B, D) from banana coal streak pathogen, banana brown gray spot pathogen, banana black spot pathogen, banana crescent spore fungus, banana alternator, banana pear spore fungus and banana anthracnose fungus, while primers NMF / NMR1 and NMF / NMR3 showed diffuse (A) and strong (C) non-specific bands when amplifying banana anthracnose fungus. It can be seen that among the four pairs of primers, NMF / NMR2 and NMF / NMR4 have better specificity.

[0053] 2.3 Sensitivity test

[0054] The concentration of genomic DNA of banana gray streak pathogen was determined by UV-visible spectrophotometer and diluted 10 times (from 200ng / μL to 1fg / μL). PCR amplification was performed using DNA of each concentration as template. The weakest band observed by naked eyes was used as the minimum detection limit. Sterile deionized water was used as negative control to test the sensitivity of 4 pairs of primers (Table 1). PCR amplification was performed at 55°C annealing temperature according to the method of 2.1 effectiveness. After the PCR reaction was completed, 5μL of the reaction product was subjected to 1% agarose gel electrophoresis. Figure 4 As shown in the figure, after electrophoresis of the amplified products of the four primer pairs in the range of 200ng / μL to 200pg / μL of genomic DNA concentration of banana gray streak pathogen, visible bands of 235bp, 268bp, 299bp and 306bp were obtained respectively (AD). Among them, primer NMF / NMR2 still had a visible band when the template was 20pg / μL (B), indicating that primer NMF / NMR2 had the highest sensitivity and the detection sensitivity could reach 20pg / μL.

[0055] Step 4: Establishment of PCR detection method for banana gray streak pathogen

[0056] 1. Extraction of genomic DNA from tissue samples to be tested

[0057] The CTAB method was used to extract the genomic DNA of the test strain and banana leaf tissue, referring to step 1 for details.

[0058] 2.PCR detection technology reaction

[0059] Based on the research results of step three, the present invention selects the primer NMF / NMR2 with strong specificity and high sensitivity as the specific amplification primer of banana ash pathogen. The extracted test strain and banana leaf tissue genomic DNA are used as templates, and the selected specific primer NMF / NMR2 is used as primer. PCR amplification is performed at an annealing temperature of 55°C. The PCR reaction system is: 12.5μLPremix Taq, 0.5μL of each 10μmol / L primer, 1μL of DNA template, and ddH2O is added to 25μL. The PCR amplification reaction conditions are: 95°C pre-denaturation for 5min; 95°C denaturation for 30sec, 55°C annealing for 30sec, 72°C extension for 45sec, a total of 30 cycles; 72°C extension for 5min; 10°C storage.

[0060] 3. PCR product electrophoresis

[0061] After the PCR reaction is completed, 5 μL of the reaction product is taken for 1% agarose gel electrophoresis analysis, and the detection result is determined according to the size of the amplified product; if a 268 bp product can be specifically amplified, it can be determined that the test sample carries banana gray streak pathogen, otherwise banana gray streak pathogen is not present in the test sample.

[0062] 4. Primer specificity verification: Amplification of banana gray streak pathogen by specific primers

[0063] (1) The CTAB method was used to extract the genomic DNA of three strains of banana gray streak pathogen, banana sooty streak pathogen, banana brown-margin gray spot pathogen, banana black spot pathogen, banana crescent spore, banana alternator spore, banana pear spore, banana anthracnose fungus, banana stick spore, and banana wilt pathogen.

[0064] (2) PCR amplification was performed using the extracted genomic DNA of the test strain as a template and sterile deionized water as a negative control. After the PCR reaction was completed, the amplified product was detected by electrophoresis.

[0065] (3) Specific amplification results

[0066] like Figure 5As shown, when the specific primers NMF / NMR2 designed and synthesized by the present invention are used as primers for PCR amplification, the three strains of banana gray streak pathogen can all specifically amplify a single band of 268 bp, while banana coal streak pathogen, banana brown gray spot pathogen, banana black spot pathogen, banana crescent spore, banana alternator spore, banana pear spore, banana anthracnose fungus, banana coryneformis, banana wilt pathogen and negative control all have no amplified fragments, indicating that the designed specific primers have relative specificity to banana gray streak pathogen, can be used to distinguish banana coal streak pathogen, banana brown gray spot pathogen, banana black spot pathogen, banana crescent spore, banana alternator spore, banana pear spore, banana anthracnose fungus, banana coryneformis, banana wilt pathogen and negative control, and have strong specificity, and can be used for specific amplification of banana gray streak pathogen.

[0067] 5. Primer sensitivity verification: Sensitivity detection of banana gray streak pathogen using specific primers

[0068] (1) The genomic DNA of banana gray streak pathogen was extracted using the CTAB method.

[0069] (2) The extracted genomic DNA of banana gray streak pathogen was measured by spectrophotometer and then diluted 10 times with sterile deionized water from 200 ng / μL to 2 fg / μL.

[0070] (3) PCR amplification is performed using the prepared DNA of a series of concentrations as a template. After the PCR reaction is completed, the amplified product is detected by electrophoresis.

[0071] (4) Sensitivity test results

[0072] like Figure 6 As shown, when the specific primers NMF / NMR2 designed and synthesized by the present invention are used for PCR, in a 25 μL reaction system, 20 pg / μL of banana gray streak pathogen DNA can obtain a visible band, and the detection sensitivity is as high as 20 pg / μL.

[0073] 6. Primer practicality verification: Pathogen detection in banana leaf tissue

[0074] (1) The CTAB method was used to extract genomic DNA from banana leaf tissue.

[0075] (2) PCR amplification was performed using the extracted genomic DNA of banana gray streak pathogen (positive control) and banana leaf genomic DNA as templates and sterile deionized water as a negative control to verify the reliability of the technology of the present invention. After the PCR reaction was completed, the amplification product was detected by electrophoresis, and the amplification product was sequenced and analyzed to confirm that the target band was a specific fragment of banana gray streak pathogen.

[0076] (3) Test results

[0077] like Figure 7 As shown, the positive control, the fresh banana leaves with natural disease in the field, the old banana leaves with natural disease in the field, the banana leaves with obvious symptoms in the indoor artificial inoculation, the banana leaves with no obvious symptoms in the indoor artificial inoculation, the banana leaves infected with banana gray stripe disease and banana coal stripe disease, the banana leaves infected with banana gray stripe disease and banana crescent spore leaf spot, the banana leaves infected with banana gray stripe disease and banana brown edge gray spot, the banana leaves infected with banana gray stripe disease and banana coryneformis leaf spot and can all produce a visible band of about 268 bp, while the healthy banana leaves and the negative control have no amplified bands. It shows that the detection method established by the present invention has good specificity and accuracy, and the primers and detection method of the present invention can be used for the detection of banana gray stripe pathogen in banana plants in the field.

[0078] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A banana gray streak pathogen Neocordana musae The PCR detection primer NMF / NMR2 is characterized in that: The upstream primer sequence was ATGTGCAAGGCCGGTTTCG, and the downstream primer sequence was CGTCACATCGGCTCAAATCG.

2. A banana gray streak pathogen Neocordana musae The PCR detection method is characterized in that The following steps are involved: (1) Extracting genomic DNA from samples to be tested; (2) Using the extracted genomic DNA of the sample to be tested as a template, PCR amplification is performed using the PCR detection primers NMF / NMR2 described in claim 1; (3) Take the PCR amplification product for electrophoresis analysis and determine the test result based on the size of the amplified product; if a 268 bp product can be specifically amplified, it can be determined that the sample carries banana gray streak pathogen. Neocordana musae Otherwise, there is no banana gray streak pathogen in the sample. Neocordana musae .

3. The PCR detection method according to claim 2, characterized in that The annealing temperature of the PCR amplification was 55°C.

4. The PCR detection method according to claim 2, characterized in that: The genomic DNA of the sample to be tested includes the genomic DNA of pathogens or the genomic DNA of banana leaves.

5. The PCR detection method according to claim 2, characterized in that: Each 25 µL PCR reaction system contains: 12.5 µL Premix Taq, 0.5 µL 10 µmol / L upstream primer, 0.5 µL 10 µmol / L downstream primer, 1 µL DNA template, and ddH2O to 25 µL.

6. The PCR detection method according to claim 2, characterized in that: The PCR amplification reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 45 sec, for a total of 30 cycles; extension at 72°C for 5 min; and storage at 10°C.

7. The PCR detection primer NMF / NMR2 according to claim 1 is effective in detecting banana gray streak pathogen Neocordana musae Application in.

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